Finding Brain Circuits Tied to Alertness

Everybody knows that it’s important to stay alert behind the wheel or while out walking on the bike path. But our ability to react appropriately to sudden dangers is influenced by whether we feel momentarily tired, distracted, or anxious. How is it that the brain can transition through such different states of consciousness while performing the same routine task, even as its basic structure and internal wiring remain unchanged?

A team of NIH-funded researchers may have found an important clue in zebrafish, a popular organism for studying how the brain works. Using a powerful new method that allowed them to find and track brain circuits tied to alertness, the researchers discovered that this mental state doesn’t work like an on/off switch. Rather, alertness involves several distinct brain circuits working together to bring the brain to attention. As shown in the video above that was taken at cellular resolution, different types of neurons (green) secrete different kinds of chemical messengers across the zebrafish brain to affect the transition to alertness. The messengers shown are: serotonin (red), acetylcholine (blue-green), and dopamine and norepinephrine (yellow).

What’s also fascinating is the researchers found that many of the same neuronal cell types and brain circuits are essential to alertness in zebrafish and mice, despite the two organisms being only distantly related. That suggests these circuits are conserved through evolution as an early fight-or-flight survival behavior essential to life, and they are therefore likely to be important for controlling alertness in people too. If correct, it would tell us where to look in the brain to learn about alertness not only while doing routine stuff but possibly for understanding dysfunctional brain states, ranging from depression to post-traumatic stress disorder (PTSD).

Though researchers have long wanted to observe in real time the transition from one brain state to another, they’ve faced stiff technical challenges. Neurons involved in neuromodulation are dispersed widely throughout the brain, and observing a transition in behavior would require scanning the entire brain at cellular resolution to track individual neuromodulatory cells mixed in among the billions of other cells. In the past, there was simply no way to determine which neurons might be firing during a transition and then to tap into their molecular information to identify them.

In the new study published in the journal Cell, Karl Deisseroth, a researcher at Stanford University, Palo Alto, CA, and colleagues found a way around this problem [1]. They turned to Danio rerio, a species of tropical freshwater fish better known in labs around the world as the zebrafish.

What sold them on zebrafish is their transparent skin, making the brain and other internal organs readily visible under a microscope. Better yet, newly hatched zebrafish are just the right size to image the entire brain, and they already react to their surroundings and show complex behaviors, such as avoiding predators or capturing prey.

First, the researchers developed a test in which they carefully immobilized the heads of the hatchlings in a standard laboratory gel while leaving their tails free to move as a sign of their mental state. They then tricked the fish into thinking a predator was present. When the perceived threat appeared, the alerted fish swished their tails to swim away.

The researchers tricked 34 hatchlings multiple times and clocked how long it took for them to swish their tails. Those hatchlings had been bioengineered so that every nerve impulse triggered a fluorescent signal, causing neurons throughout the brain to light up with color while they were in the gel. As a result, the researchers were able to capture the activity of up to 35,000 neurons throughout the animals’ brains with a custom-designed microscope each time they tried to swim away. What the researchers were most interested in was the signals in the brain observed when the animals were most alert, as evidenced by their quick reaction times.

Deisseroth’s team then preserved the animals’ brain tissue for further analysis. This enabled the researchers to go back and use fluorescently tagged antibodies and a high-powered microscope to track the activation of specific neurons when the hatchlings were most alert. They call their new two-step method Multi-MAP, short for Multiplexed-alignment of Molecular and Activity Phenotypes.

This innovative method pointed them to multiple distinct types of neurons that coordinated their activities in a distinctive pattern when the fish transitioned to alertness. For example, the identified a group of neurons that secrete the stress-response chemical norepinephrine in the locus coeruleus, an area of the brain that’s previously been tied to alertness. The study also turned up some groups of neurons that appear to secrete the chemical nerve messengers acetylcholine, serotonin, and dopamine, which adjust the sensitivity of other neurons.

This brought researchers to the bigger question. Would the findings in zebrafish apply to other animals? To find out, they used another method to record the activity of those same neural cell types in the brains of mice. The mice had been trained to lick upon hearing a stimulating sound that perked them to attention. Those studies confirmed a similar pattern of coordinated activity in the brains of more alert mice.

The researchers next used optogenetics—a groundbreaking technology also pioneered by Deisseroth that makes it possible to activate neurons with light—to confirm in the mice a causal connection between activity in the identified neural cell types and an alert state. Those studies narrowed the field to three groups of neurons in different parts of the brain that appear sufficient to increase alertness when activated and now warrant further investigation.

The new findings open a whole new vista for exploring how these different brain areas work together to increase alertness. Deisseroth says he and his team now hope to apply their new Multi-Map technique to explore other brain states, such as PTSD and depression, that are characterized by increases or decreases in alertness. Now, that’s something to ponder next time you’re out trying to stay alert while walking on the bike path.

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About the NIH Director

Francis S. Collins, M.D., Ph.D.

Appointed the 16th Director of NIH by President Barack Obama and confirmed by the Senate. He was sworn in on August 17, 2009. On June 6, 2017. President Donald Trump announced his selection of Dr. Collins to continue to serve as the NIH Director.